Anti-inflammatory, tolerogenic and immunoinhibiting properties of carbohydrate binding-peptides

ABSTRACT

The present invention is directed to methods of suppressing inflammatory responses, including tolerance to an antigen, and suppressing cell adhesion, e.g., involved in metastasis, by the administration of lectin derived carbohydrate binding peptides or derivatives thereof, in particular, peptides capable of binding terminally linked α-sialic acid (2→6)βGal- and/or α-sialic acid(2→3)βGal-groups on structures or molecules comprising such groups. Pharmaceutical compositions containing such lectin derived carbohydrate binding peptides or derivatives thereof are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. Ser. No. 07/995,503 filed Dec.21, 1992, now U.S. Pat. No. 5,453,272, which, in turn, is acontinuation-in-part of U.S. Ser. No. 07/956,043, filed Oct. 2, 1992,now abandoned, which application is incorporated herein by reference inits entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to methods for inhibiting immune responses orcellular interactions in mammals by the administration thereto of one ormore lectin derived carbohydrate binding peptides. In particular, thisinvention is directed to methods for the suppression of inflammatoryresponses, induction of tolerance to antigens, modulation of theinduction of immune responses to antigens, and the inhibition of celladhesion in mammals by the administration of one or more carbohydratebinding peptides. The lectin derived carbohydrate binding peptidesemployed herein are preferably fragments of the S2 or S3 subunits of thepertussis toxin expressed by Bordetelia pertussis or functionallyequivalent variants thereof.

2. References

The following references are cited in this application as superscriptnumbers at the relevant portion of the application:

1. Brandley, et al., J. Leukocyte Biol., 40:97-111 (1986).

2. Jacobson, Developmental Neurobiology, New York, Plenum Press p. 5-25,(1978).

3. Trinkaus, Cells into Organs, Englewood Cliffs, N.J., Prentice Hall,p. 44-68, (1984).

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6. Paulson, In "The Receptors", Vol. II (Comm., P.M., Ed.), New YorkAcademic Press, p. 131 (1985).

7. Sharon, Lectin-Like Bacterial Adherence to Animal Cells. In"Attachment of Microorganisms to the Gut Mucosa" (Boeheker, E. D., Ed.),Boca Raton, Fla. CRC Press, p. 129 (1984).

8. Wassarman, Fertilization. In "Cell Interactions and Development:Molecular Mechanisms" (Yamada, K. M., Ed.), New York, John Wiley andSons, p. 1 (1983).

9. Schwartz, et al., Immunol. Rev., 40:153 et seq. (1978).

10. Coutinho, et al., Immunol. Rev., 78:211 et seq. (1984).

11. Hoffmann, et al., Eds., Membranes in Growth and Development, NewYork, Alan R. Liss, p. 429-442, (1982).

12. Galeotti, et al., Eds., Membranes in Tumor Growth, Amsterdam,Elsevier, p. 77-81, (1982).

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14. Aplin, et al., Biochim. Biophys. Acta 694:375 et seq. (1982).

15. Barondes, Developmentally Regulated Lectins. In "Cell Interactionsand Development: Molecular Mechanisms" (Yamada, D. M., Ed.) New York,John Wiley and Sons, p. 185 (1983).

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22. Bevilacqua, et al., Endothelial-Leukocyte Adhesion Molecule-1(ELAM-1): A Vascular SELECTIN That Regulates Inflammation. In "Cellularand Molecular Mechanisms of Inflammation" Vol. 2, Academic Press, p.1-13 (1991).

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The disclosure of all publications, patents and patent applicationscited in this application are herein incorporated by reference in theirentirety to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety.

3. State of the Art

Important processes involving mammalian cells, such as growth,locomotion, morphological development, and differentiation are partiallycontrolled by extracellular signals acting upon the cells' surfaces¹⁻³.While some external stimuli reach the cell via extracellular fluids,other signals are received from neighboring or approaching cell surfacesand exert their effects through direct cell-cell contact⁴,5.

Evidence suggests that specific cell-surface receptors can "sense" amolecular signal of an apposing cell via specific binding, andbiochemical mechanisms exist to translate that binding into a cellularresponse. For example, complex cell-surface interactions are believed tohelp direct processes such as binding of pathogens to target tissues⁶,7,sperm-egg binding⁸, interactions among cells in the immune system⁹,10,and recognition of cells during embryonic development¹¹. In addition,defects in cell-cell recognition are thought to underlie theuncontrolled cell growth and motility which characterize neoplastictransformation and metastasis¹²,13.

Other evidence suggests that cell-recognition processes are mediated bycarbohydrate chains or glycan portions of glycoconjugates⁴,14-16. Forexample, the binding of the surface glycoconjugates of one cell to thecomplementary carbohydrate-binding proteins (lectins) on another cellcan result in the initiation of a specific interaction.

One important group of carbohydrate-binding proteins are selectin(LEC-CAM) proteins (Lectin+EGF +complementary Regulatory Domain-CellAdhesion Molecules). These or functionally similar proteins or lectinsare believed to play a critical role in immune responses (includinginflammatory responses) through mediation of cell-cell contact andthrough extra-vasation of leucocytes¹⁷⁻²². Specific carbohydrate ligandshave been identified as part of the putative receptor structures forselectin proteins and other lectins¹⁷⁻²⁵. The structures identifiedinclude oligosaccharide glycosides containing terminally linked α-sialicacid(2→6)βGal- and α-sialic acid(2→3)βGal-groups. The use ofoligosaccharides and derivatives thereof having such terminally linkedgroups for controlling inflammation, immunosuppression, etc. byinteracting with selectin proteins and/or other lectins has beendisclosed²⁶⁻²⁹,35-41.

Likewise, peptides derived from the selectin GMP-140 which inhibitbinding of GMP-140 and other selectins to leukocytes, presumably byinterfering with the binding of the GMP-selectin protein with thecarbohydrate receptor on the leukocyte, have also been disclosed asbeing useful in suppressing an immune response²⁹. Similarly, otherpeptides are also known to be potent modulators of neutrophilfunctions⁴⁸.

Pertussis toxin (PT)⁴², a virulence factor produced by the organismBordetella pertussis--the etiological agent of whooping cough, is knownto bind to glycoprotein receptors which terminate in sialyllactosaminesugar sequences⁴³,44 and we have previously shown that this protein isuseful in suppressing mammalian immune responses and cellularadhesion⁴⁵. PT's binding specificity has been shown to be similar tothat of the plant lectins from Sambucus nigra (SNA) and Maackiaamurensis (MAL)²⁵, which bind with high affinity to sialicacid-containing glycoconjugates⁴⁶,47 and which have also been shown aspossessing immunomodulating properties⁴⁵.

However, the use of proteins or large molecular weight polypeptides ininhibiting immune responses or cellular interactions in mammals suffersfrom several drawbacks, including the fact that they are difficult toproduce in large quantities in pure form; that they tend to produceadverse affects when repeatedly administered to a mammal; that they cancontain infectious agents or toxic substances which arecontra-indications to mammalian administration; and, that it isdifficult to modify the pharmokinetic properties of such proteins toimprove their efficacy.

In view of the above, the use of peptides having lectin-like bindingproperties for terminal αNeu5Ac(2→3)βGal- and αNeu5Ac(2→6)βGal- groupswould be particularly beneficial for use in inhibiting immune responsesand cellular interactions in mammals as compared to the administrationof proteins such the pertussis toxin and the lectins derived from SNAand MAL because such peptides would mitigate the problems associatedwith the administration of proteins and large molecular weightpolypeptides to mammals.

SUMMARY OF THE INVENTION

This invention is directed, in part, to the discovery that the bindingdomains for the α-sialic acid(2→3)βGal- and/or the α-sialicacid(2→6)βGal-terminally linked structures are found in certain peptidefragments of lectins (e.g., proteins and polypeptides) such as pertussistoxin and that it is not necessary to employ the entire lectin to effectbinding to these carbohydrates (oligosaccharides).

This invention is further directed, in part, to the discovery that, whenthese lectin derived carbohydrate binding peptides are administered to amammal (e.g., human) in effective amounts, they inhibit specific immuneresponses and cellular interactions. In particular, this invention isdirected to the discovery that such lectin derived carbohydrate bindingpeptides may be administered to a mammal in order to inhibitinflammatory responses, modulate the induction of an immune response toan antigen, induce long term tolerance to an antigen, and suppress celladhesion.

This invention is particularly directed to the discovery that lectinderived carbohydrate binding peptides capable of binding terminallylinked α-sialic acid(2→3)βGal- and/or α-sialic acid(2→6)βGal- (e.g.,αNeu5Ac(2→3)βGal-) groups present in molecules (e.g., oligosaccharides,glycoproteins, glycolipids, etc.) which can be found on cell surfaces(e.g., leukocytes) may be administered to a mammal as a means forinhibiting cell adhesion or cell-mediated immune responses.Cell-mediated immune responses inhibited by the peptides disclosedherein include inflammatory responses, modulation of the induction ofthe immune response to an antigen and the induction of long termtolerance to an antigen.

Preferably, the lectin derived carbohydrate binding peptides employedherein are fragments of the S2 or S3 subunits of the pertussis toxinexpressed by Bordetella pertussis or functionally equivalent variantsthereof. These preferred peptides have the amino acid sequences setforth in peptides of FIG. 1 below.

Accordingly, in one of its method aspects, this invention is directed toa method of suppressing a inflammatory response in a mammal by theadministration of an effective amount of at least one lectin derivedcarbohydrate binding peptide or derivative thereof capable of binding aterminally linked α-sialic acid(2→6)βGal- and/or α-sialic acid(2→3)βGal-groups on structures or molecules comprising such groups.

In another of its method aspects, this invention is directed to a methodfor modulating the induction of an immune response to an antigen in amammal by administering the antigen in combination with an effectiveamount of at least one lectin derived carbohydrate binding peptide orderivative thereof capable of binding terminally linked α-sialicacid(2→6)βGal- and/or α-sialic acid(2→3)βGal- groups on structures ormolecules comprising such groups.

In another of its method aspects, this invention is directed to a methodfor inducing in a sensitized mammal long term tolerance to an antigen byexposing (challenging) the mammal with the antigen followed by theadministration of an effective amount of at least one lectin derivedcarbohydrate binding peptide or derivative thereof capable of bindingterminally linked α-sialic acid(2→6)βGal- and/or α-sialic acid(2→3)βGal-groups on structures or molecules comprising such groups.

In still another one of its method aspects, this invention is directedto a method for inhibiting cell adhesion events involved in metastasisof tumor cells or in inflammation by the administration of an effectiveamount of at least one lectin derived carbohydrate binding peptide orderivative thereof capable of binding terminally linked α-sialicacid(2→6)βGal- and/or α-sialic acid(2→3)βGal- groups on structures ormolecules comprising such groups.

In yet another one of its method aspects, this invention is directed toa method for treating lung inflammation and/or lung injury in a mammalby the administration of an effective amount of one or more lectinderived carbohydrate binding peptides or derivative thereof capable ofbinding terminally linked α-sialic acid(2→6)βGal- and/or α-sialicacid(2→3)βGal-groups on structures or molecules comprising such groups.

In its composition aspects, this invention is directed to pharmaceuticalcompositions comprising the subject lectin derived carbohydrate bindingpeptides including pharmaceutically acceptable salts of such lectinderived carbohydrate binding peptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the amino acid sequence of preferred lectin derivedcarbohydrate binding peptides.

FIG. 2 illustrates the increase in footpad swelling of immunized Balb/cmice arising from a DTH inflammatory response measured 24 hours afterchallenge with 20 μg of OVA antigen wherein some of the mice have beentreated at 5 hours after challenge with 100 μg of a lectin derivedcarbohydrate binding peptide.

FIG. 3 illustrates the long term (2 week) effects on the DTH responsesdetermined by footpad swelling, in groups of Balb/c mice which areimmunized with 100 μg of the OVA antigen, challenged 7 days later withOVA, and then treated about 5 hours thereafter with a lectin derivedcarbohydrate binding peptide.

In FIGS. 2 and 3, peptide 2275 is ACS2P1 (amino acids 9-23 of SEQ ID NO:3) and peptide 2283 is SPYGRC (amino acids 18-23 of SEQ ID NO:3) both ofwhich are illustrated in FIG. 1.

The amino acid residues depicted in FIG. 1 and used throughout thisapplication are designated by the conventional single letter code setforth below:

    ______________________________________                                        A      alanine     I      isoleucine                                                                            R    arginine                               C      cystine     K      lysine  S    serine                                 D      aspartic acid                                                                             L      leucine T    threonine                              E      glutamic acid                                                                             M      methionine                                                                            V    valine                                 F      phenyl alanine                                                                            N      asparagine                                                                            W    tryptophane                            G      glycine     P      proline Y    tyrosine                               H      histidine   Q      glutamine                                           ______________________________________                                    

Additionally, as conventionally indicated, the amino acid residueforming the H₂ N- terminus of the peptide is set forth on the left sideof the peptide chain and the --COOH terminus of the peptide is set forthon the right side of the peptide chain.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention is directed, in part, to the discovery that certainlectin derived carbohydrate binding peptides when administered to amammal are effective in suppressing inflammatory responses, inducingtolerance to an antigen, modulating the induction of immune responses toantigens, and inhibiting cell adhesion events, e.g., cell adhesionevents involved in metastasis of tumor cells.

1. Definitions

As used herein, the following terms have the meanings set forth below:

The term "inflammatory response" or "inflammatory disorder" will referto immune reactions involving specific and non-specific defense systems.A specific defense system reaction is a specific immune system reactionto an antigen. Examples of specific defense system reactions includeantibody responses to antigens, such as viruses, allergens, anddelayed-type hypersensitivity. A non-specific defense system reaction isan inflammatory response mediated by leukocytes generally incapable ofimmunological memory. Such cells include macrophages, eosinophils andneutrophils. Examples of non-specific reactions include the immediateswelling after a bee sting, and the collection of polymorphonuclear(PMN) leukocytes at sites of bacterial infection (e.g., pulmonaryinfiltrates in bacterial pneumonias and pus formation in abscesses).

Other "inflammatory responses" or "inflammatory disorders" within thescope of this invention include, e.g., autoimmune disorders such asrheumatoid arthritis, lupus, multiple sclerosis, post-ischemic leukocytemediated tissue damage (reperfusion injury), frost-bite injury or shock,acute leukocyte-mediated lung injury (ARDS), asthma, traumatic shock,septic shock, nephritis, and acute and chronic inflammation, includingatopic dermatitis, psoriasis, and inflammatory bowel disease. Variousplatelet-mediated pathologies such as atherosclerosis and clotting arealso included within the definition of "inflammatory responses" or"inflammatory disorders". In addition, "inflammatory responses" or"inflammatory disorders" may include the adhesion of circulating cancercells, with specific examples including carcinoma of the colon andmelanoma.

Without being limited to any theory, we believe that primary events inthe initiation of such inflammatory responses and inflammatory disordersare the binding of leukocytes to selectins (e.g., ELAM-1, PADGEM, etc.)through carbohydrate receptors comprising α-sialic acid(2→3)βGal- and/orα-sialic acid(2→6)βGal-groups³⁵⁻⁴⁰ found on the surface of theleukocytes. Likewise, it has been shown that mammalian and, inparticular, human cancer cells contain α-sialic acid(2→3)βGal- and/orα-sialic acid(2→6)βGal- groups on the surface thereof⁴⁹,50 and it isbelieved that binding of such circulating cancer cells to selectins isan integral part of the metastatic process³⁶,37. Accordingly, byinterfering with the binding of such carbohydrate receptors to theseselectins, suppression of the inflammatory immune responses as well asinhibition of metastatic processes is achieved.

The term "antigen" refers to any protein, peptide, carbohydrate, nucleicacid or other non-endogenous substance which when exposed to a mammalinduces an immune response in that mammal.

Disease conditions believed to be caused by antigen exposure include, byway of example, psoriasis, asthma, dermatitis, rheumatoid arthritis,delayed type hypersensitivity, inflammatory bowel disease, multiplesclerosis, viral pneumonia, bacterial pneumonia, and the like.

The term "non-sensitized mammal" refers to those mammals which have yetto be educated to a particular antigen.

The term "sensitized mammal" refers to those mammals which have beenpreviously exposed to a particular antigen and, accordingly, theirimmune systems have become educated to that antigen. Typically, initialexposure of an antigen to a mammal primes or educates the mammal'simmune response to later exposure to that antigen with minimalinflammation during such initial exposure.

The term "secondary immune response" refers to the effector phase of amammal's immune response to an antigen to which it has been previouslybeen sensitized. A mammal's secondary immune response is typicallyaccompanied by inflammation at the point of antigen exposure.

"Acute respiratory distress syndrome" or "ARDS" refers to aninflammatory condition comprising leukocyte mediated lung injury.Without being limited to any theory, it is believed that such lunginjury is exacerbated by infiltration and subsequent disruption ofneutrophils into the lungs. Specifically, the disruption of theneutrophils in the lungs releases superoxides which results in severevascular endothelial damage. Accordingly, while this lung damage is notantigen based, the infiltration of neutrophils into the lungs requiresan adhesion event.

"Reperfusion injury" refers to an inflammatory condition comprisingleukocyte mediated tissue damage. Reperfusion injury commonly occursafter myocardial infarction wherein, in response to the inflammationcaused by the myocardial infarction, the endothelium cells are activatedand produce selectins (e.g., ELAM-1). In turn, neutrophils are thencapable of binding the selectins expressed on the vascular endotheliumand cause further damage.

The term "pertussis toxin" or "PT" in this application refers to thevirulence factors produced by Bordetella pertussis, the etiologicalagent of whooping cough. The β-oligomer of PT binds to both α-sialicacid(2→6)βGal- and α-sialic acid(2→3)βGal- structures. PT also hassimilar binding characteristics to those of wheat germ agglutinin (WGA),which can recognize terminal N-acetylglucosamine (GlcNAc) saccharidesequences in addition to sialic acid⁴⁴,52,53.

PT is a classical A-B type of toxin comprised of an A subunit(designated S1) that contains an ADP-ribosyltransferase enzyme activity,which is responsible for most of the biological effects of PT⁵⁴. Thelectin-like activity is found in the complex β oligomer of PT, whichconsists of four heterogeneous subunits that are arranged in a pair ofdimers (S2-S4 (Dimer 1) and S3-S4 (Dimer 2)) joined by a smaller S5subunit. The functioning of the β oligomer is in binding to host cellsialylated oligosaccharide receptors as well as providing a deliverysystem for the A subunit through the cytoplasmic membrane⁵⁴,55,56. The βoligomer itself can induce a mitogenic response in lymphocytes and hasthe ability to agglutinate erythrocytes. PT may also contribute to theattachment of B. pertussis to epithelial cells lining the upperrespiratory tract of humans; the only known host of B. pertussis⁵⁷. Inaddition, the β oligomer also appears to share functional homology withthe selectin family of mammalian lectins that regulate leukocytetrafficking.⁵⁸

The term "cell-mediated immune response" refers to those mammalianimmune responses which are mediated by cell-cell interactions. Includedwithin this term are cell-mediated inflammatory responses to an antigenincluding, by way of example, such responses as delayed-typehypersensitivity (DTH) responses, virus-induced pneumonia, allergicresponses, and the like as well as cell-mediated inflammatory responsesarising from injuries such myocardial infarction, shock and sequelae(e.g., multiple organ failure), acute respiratory distress syndrome,(ARDS), and the like. Generally, the cell-mediated immune response is aleukocyte-mediated response.

The term "humoral immune response" refers to mammalian immune responseswhich involve antigen-antibody interactions.

The term "DTH inflammation response" or delayed type hypersensitivityresponse is a T cell mediated reaction which results in a mononuclearcell-rich inflammation and swelling which occurs after antigenicchallenge.

The term "tolerance" or "immunological tolerance" refers to a reducedimmunogenic response elicited in a sensitized mammal to a particularantigen upon a second or subsequent antigenic challenge in comparison tothe primary immune response elicited by said antigen under equivalentconditions (e.g., dosage). In the present invention such "tolerance"will be obtained by administration of an antigen to the sensitizedmammal followed by administration of one or more lectin derivedcarbohydrate binding peptides which bind to α-sialic acid(2→6)βGal-and/or α-sialic acid(2→3)βGal- structures.

The term "period for maximal inflammation" refers to the period of timetypically required to achieve maximal inflammation in a mammal due to acell-mediated immune response including both cell-mediated inflammatoryresponses in a sensitized mammal due to antigen exposure (challenge) andcell-mediated inflammatory responses in a mammal due to injury (e.g.,myocardial infarction). This period of time depends on several factorssuch as the specific antigen/injury afflicting the mammal; theparticular mammalian species exposed to the antigen/injury, etc.Accordingly, the period of time required to effect maximal inflammationwill vary for, by way of example, rheumatoid arthritis as opposed tomyocardial infarction.

Moreover, while the specific time required to effect maximalinflammation will vary somewhat in a given mammalian species, the timetypically required to effect maximal inflammation for differentafflictions due to either antigen exposure or injury in human and othermammals is known in the art or are readily ascertainable by the skilledartisan. For example, in the case of a DTH response in mice, maximalinflammation is typically 24 hours after antigen exposure.

The term "sialic acid" refers to all naturally occurring structures ofsialic acid and analogues of sialic acid as well as derivatives thereof.Naturally occurring structures of sialic acid include, by way ofexample, 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulopyranosylonicacid ("Neu5Ac"), N-glycoyl neuraminic acid (Neu5Gc) and 9-O-acetylneuraminic acid (Neu5,9Ac₂). A complete list of naturally occurringsialic acids known to date are provided by Schauer⁵¹.

Derivatives of sialic acid refers to derivatives of naturally occurringstructures of sialic acid including those wherein the sialic acid unithas been chemically modified so as to introduce and/or remove one ormore functionalities from such structures. For example, suchmodification can result in the removal of an --OH functionality, theintroduction of an amine functionality, the introduction of a halidefunctionality, and the like.

Certain derivatives of sialic acid are known in the art and includechemically modified sialic acid derivatives such as 9-azido-Neu5Ac,9-amino-Neu5Ac, 9-deoxy-Neu5Ac, 9-fluoro-Neu5Ac, 9-bromo-Neu5Ac,8-deoxy-Neu5Ac, 8-epi-Neu5Ac, 7-deoxy-Neu5Ac, 7-epi-Neu5Ac,7-8-bis-epi-Neu5Ac, 4-O-methyl-Neu5Ac, 4-N-acetyl-Neu5Ac,4,7-di-deoxy-Neu5Ac, 4-uno-Neu5Ac, 3-hydroxy-Neu5Ac, 3-fluoro-Neu5Acacid as well as 6-thio analogues of Neu5Ac are known in the art. Methodsfor preparing such sialic acid derivatives are taught in commonlyassigned Docket No. 000475-005, U.S. Ser. No. 07/889,017, filed on May26, 1992, which application is incorporated by reference in itsentirety.

The nomenclature describing derivatives of sialic acid derivativesherein is as set forth by Reuter et al³⁴.

The term "α-sialic acid(2→6)βGal- structures or groups" refer tomolecules comprising the terminally linked α-sialic acid(2→6)galactose-sequence or derivatives thereof. Molecules containing such terminalstructures have been identified as comprising part of the putativereceptor structure for the ELAM-1 and PADGEM selectins³⁸,40.

The term "α-sialic acid(2→3)βGal- structures or groups" refer tomolecules comprising the terminally linked α-sialic acid(2→3)galactose-sequence or derivatives thereof. Molecules comprising such terminalstructures have similarly been identified as comprising part of theputative receptor structures for the ELAM-1 selectins³⁵⁻³⁹.

The term "lectin derived carbohydrate binding peptide" refers to anypeptide or derivative thereof (including pharmaceutically acceptablesalts) derived from a lectin and which is capable of binding to α-sialicacid(2→6)βGal- and/or α-sialic acid(2→3)βGal-carbohydrate structureswhich are preferably comprised on the surface of mammalian cells.Generally, in the present application, "lectin derived carbohydratebinding peptide" will refer to such peptides which, in monomeric form,have no more than about 35 amino acids in the lectin-like domain (i.e.,the part of the peptide responsible for binding to such carbohydratestructures). Suitable derivatives of lectin derived carbohydrate bindingpeptides include those peptides which have the NH₂ and/or the COOHterminal functionalities blocked by conventionally blocking groups aswell as derivatives which include modifications, deletions orderivatizations of one or more of the amino acids that yield a peptidewhich is capable of binding to α-sialic acid(2→6)βGal- and/or α-sialicacid(2→3)βGal- structures.

More preferably, the lectin derived carbohydrate binding peptides referto the peptides set forth in FIG. 1.

Still more preferably, the lectin derived carbohydrate binding peptidesare peptides which have a high degree of homology with a lectin-likebinding domain for the α-sialic acid(2→6)βGal- and/or α-sialicacid(2→3)βGal- carbohydrate structures found in the pertussis toxin.These peptides are represented by formula I (SEQ ID NO:1):

    SPX.sub.1 GX.sub.2 C                                       I

where X₁ is selected from the group of amino acids Y, F, W, and H orpeptide mimetics thereof, and X₂ is selected from the group consistingof amino acids Y, F, R, W, and H or peptide mimetics thereof;

or by formula II (SEQ ID NO:2):

    SPX.sub.1 GX.sub.2 CX.sub.3 X.sub.4                        II

where X₁ is selected from the group of amino acids Y, F, W, and H orpeptide mimetics thereof,

X₂ is selected from the group consisting of amino acids Y, F, R, W, andH or peptide mimetics thereof;

X₃ is an amino acid sequence of 4-6 amino acids; and

X₄ is selected from the group consisting of amino acids Y, F, W, and Hor peptide mimetics thereof.

Most preferably, the lectin derived carbohydrate binding peptide refersto the hexapeptide SPYGRC (amino acids 18-23 of SEQ ID NO:3).

In addition to their use as modulating agents for mammalian immune andcellular adhesion processes, the lectin derived carbohydrate bindingpeptides described herein can further be used in assay methods fordetermining the presence of α-sialic acid(2→3)βGal- and/or α-sialicacid(2→6)βGal- structures on molecules and/or cell surfaces such as insuspected cancer cells; for determining the relative binding affinity ofpeptides and/or proteins to α-sialic acid(2→3)βGal- and/or α-sialicacid(2→6)βGal- structures; and the like. When so employed, the lectinderived carbohydrate binding peptides typically will be derivatized toinclude a label or a label binding moiety.

Suitable labels are well known in the art and include, by way ofexample, enzymes (e.g., horseradish peroxidase), radioisotopes (e.g.,¹²⁵ I), fluorescent moieties, chemiluminscent moieties, and the like.The particular label employed is not critical and methods for attachinglabels to peptides are well known in the art.

Suitable label binding moieties are also well known in the art andinclude, by way of example, biotin, avidin, streptavidin antibodies,etc. A preferred label binding moiety is biotin which permits binding ofup to 4 peptide/biotin adducts to avidin. The avidin can beappropriately labeled so that resulting peptide/biotin/avidin complexcan be detected.

The term "lectins" refer to carbohydrate binding proteins of non-immuneorigin often obtained from plants or bacterial or viral microorganismswhich comprise carbohydrate binding sites. These binding proteinstypically comprise the ability to agglutinate cells and to precipitatecomplex carbohydrates. Lectins are classified based upon theircarbohydrate binding specificity and are well known in the art.

The term "lectin-like domain" refers to those fragment(s) of a lectinresponsible for binding the carbohydrate.

The term "pharmaceutically acceptable salts" includes thepharmaceutically acceptable addition salts of lectin derivedcarbohydrate binding peptides capable of binding to terminally linkedα-sialic acid(2→6)βGal- and/or α-sialic acid(2→3)βGal-. Suchpharmaceutically acceptable addition salts may be derived from a varietyof organic and inorganic counter salts well known in the art andinclude, by way of example only, sodium, potassium, calcium, magnesium,ammonium, tetralkyl-ammonium, and the like.

2. Utility

Without being limited to any theory, it is believed that the subjectlectin derived carbohydrate binding peptides affect the immune responsein a number of ways. Lectin derived carbohydrate binding peptides caninhibit a mammal from becoming "educated" about a specific antigen whenthe lectin derived carbohydrate binding peptide is administeredsimultaneously with the first exposure of the immune system to theantigen.

The lectin derived carbohydrate binding peptides can reducecell-mediated immune responses to injury such as inflammatory responsesarising from myocardial infarction, ARDS, frost bite, etc. The lectinderived carbohydrate binding peptides can also inhibit the effectorphase of a cell-mediated immune response (e.g., the inflammatorycomponent of a DTH response) when administered to a sensitized mammalafter exposure of the sensitized mammal's immune system to the antigen.In either case, in order to effect reduction in the cell-mediated immuneresponse, it is necessary to administer the lectin derived carbohydratebinding peptides after initiation of the mammal's immune response and ator prior to one-half the period required for maximal inflammationinduced by the injury or the antigen exposure.

Additionally, the subject lectin derived carbohydrate binding peptidescan induce tolerance to antigens in sensitized mammals when administeredat the time of second or later exposures of the immune system to theantigen when administration is conducted after initiation of themammal's secondary immune response to the antigen and at or prior toone-half the period required for maximal inflammation induced by theantigen exposure.

Further, the administration of lectin derived carbohydrate bindingpeptides that bind α-sialic acid(2→6)βGal- and/or α-sialicacid(2→3)βGal-structures inhibit the binding of LEC-CAM proteins andother selectins to their putative receptors therefor which include boththe α-sialic acid(2→6)βGal-³⁸,40 and the α-sialic acid(2→3)βGal-³⁵⁻³⁹structures.

Accordingly, the subject invention provides both pharmaceuticalcompositions containing lectin derived carbohydrate binding peptidescapable of binding terminally linked α-sialic acid(2→6)βGal- and theα-sialic acid(2→3)βGal-structures which are useful in inhibitingspecific immune responses or cellular interactions in mammals as well asmethods which include the administration of such lectin derivedcarbohydrate binding peptides to a mammal for inhibiting a cell-mediatedimmune response.

As noted above, lectin derived carbohydrate binding peptides useful formodulating a cell-mediated immune response in a mammal include anylectin derived peptide or derivative thereof capable of bindingterminally linked α-sialic acid(2→6)βGal- and the α-sialicacid(2→3)βGal-structures. Suitable lectin derived carbohydrate bindingpeptides preferably are peptides which, in monomeric form, have no morethan about 35 amino acids in the lectin-like domain (i.e., the part ofthe peptide responsible for binding to such carbohydrate structures).

More preferably, the lectin derived carbohydrate binding peptides referto the peptides set forth in FIG. 1.

Still more preferably, the lectin derived carbohydrate binding peptidesare peptides which have a high degree of homology with a lectin-likebinding domain for terminally linked α-sialic acid(2→6)βGal- and/orα-sialic acid(2→3)βGal- carbohydrate structures found in the pertussistoxin. These peptides are represented by formula I (SEQ ID NO:1):

    SPX.sub.1 GX.sub.2 C                                       I

where X₁ is selected from the group of amino acids Y, F, W, and H orpeptide mimetics thereof, and X₂ is selected from the group consistingof amino acids Y, F, R, W, and H or peptide mimetics thereof;

or by formula II (SEQ ID NO:2):

    SPX.sub.1 GX.sub.2 CX.sub.3 X.sub.4                        II

where X₁ is selected from the group of amino acids Y, F, W, and H orpeptide mimetics thereof,

X₂ is selected from the group consisting of amino acids Y, F, R, W, andH or peptide mimetics thereof;

X₃ is an amino acid sequence of 4-6 amino acids; and

X₄ is selected from the group consisting of amino acids Y, F, W, and Hor peptide mimetics thereof.

The preparation of such peptides is well known in the art and includes,by way of example, standardized commercially available peptidesynthesizers such as Model ABI 403A available from Applied Biosystems,Inc., Foster City, Calif. The methods for preparing such peptides doesnot form a part of this invention.

Peptide mimetics refer to groups which mimic an amino acid in thepeptide chain. Such mimetics and their synthesis are well known in theart⁵⁹.

The lectin derived carbohydrate binding peptides can be used either inmonomeric or polymeric form. Examples of suitable polymers include theattachment of biotin to the lectin derived carbohydrate binding peptidefollowed by complexing with avidin which results in up to a tetravalentcomplex. Likewise, multivalent derivatives of lectin derivedcarbohydrate binding peptides can be synthesized by attaching suchpeptides to polymers such as polylysine or an inert protein such ashuman serum albumin. The multivalent derivatives so formed can containone or a mixture of different lectin derived carbohydrate bindingpeptides so as to enhance efficacy.

In the case of protein peptide conjugates, they will be chemicallycross-linked with the carrier protein by known cross-linking agentsusing art recognized methodology.

In yet another embodiment, multivalent lectin derived carbohydratebinding peptides can be generated as a copolymer wherein the peptidesare linked together through a spacer arm to provide for a repeatingsubunit represented by the groups:

     carbohydrate binding peptide-spacer arm!.sub.n

     carbohydrate binding peptide!.sub.n -spacer arm

In these subunits, the lectin derived carbohydrate binding peptide canbe the same or different lectin derived carbohydrate binding peptide andthe spacer arm is selected to provide for optimal distance to bind tothe carbohydrate.

However, the invention is not restricted to the use of lectin derivedcarbohydrate binding peptides specifically exemplified in FIG. 1 or tomultivalent derivatives thereof, but rather embraces the use of anylectin derived peptide or derivative thereof which binds terminallylinked α-sialic acid(2→6)βGal- and/or α-sialic acid(2→3)βGal-carbohydrate structures. As noted previously, peptides capable ofbinding such structures, when administered to a mammal, result in theinhibition of immune responses and cellular interactions, in particular,inflammatory responses or conditions, tolerance to antigens, modulationof the immunogenic response to antigens, and the inhibition of celladhesion events, which are involved, e.g., in metastasis andinflammation.

It is well within the level of ordinary skill to identify other lectinderived peptides capable of binding terminally linked α-sialicacid(2→6)βGal- and/or α-sialic acid(2→3)βGal- structures, byconventional methods for assaying binding between ligands. Such methodsinclude, e.g., competitive binding assays and receptor binding assays.The subject application, in particular, sets forth one method in theexamples which illustrates rather simple assaying techniques that arecapable of determining binding to terminally linked α-sialicacid(2→6)βGal- and/or α-sialic acid(2→3)βGal- structures. Other methodsfor determining the binding of a candidate peptide with such terminallylinked structures are known in the art. See, for example, Pearce-Prattet al³¹.

The subject invention accordingly further provides a method by whichlectin derived peptides capable of inducing or suppressing variousimmune responses and cellular interactions, e.g., inflammation,antigenic tolerance, modulation of antigenic response, or cell adhesion,may be putatively identified on the basis of their ability to bind toterminally linked α-sialic acid(2→6)βGal- and/or sialic acid(2→3)βGal-structures.

In regard to the above, the subject invention contemplates theattachment of labels or label binding groups to the lectin derivedcarbohydrate binding peptides and/or to candidate lectin derivedcarbohydrate binding peptides in order to facilitate the assaysdescribed above. Such labels are conventionally formed on the peptidesby methods well known in the art. Suitable labels include, by way ofexample, enzymes (e.g., horseradish peroxidase), radioisotopes (e.g.,¹²⁵ I), fluorescent moieties, chemiluminscent moieties, and the like.

Suitable label binding moieties are also well known in the art andinclude, by way of example, biotin, avidin, antibodies, etc. A preferredlabel binding moiety is biotin which permits binding of thepeptide/biotin adduct to avidin. The avidin can be appropriately labeledso that resulting peptide/biotin/avidin complex can be detected.

The subject invention also contemplates kits for use in conducting suchassays. Such kits would comprise the labelled lectin derivedcarbohydrate binding peptide or the lectin derived carbohydrate bindingpeptide attached to label binding groups.

Suitable lectin derived carbohydrate binding peptides for use herein arethose which are capable of binding terminally linked α-sialicacid(2→6)βGal- and/or α-sialic acid(2→3)βGal- structures. However, anadditional prerequisite of efficacious lectin derived carbohydratebinding peptides will include suitability for in vivo administration. Inparticular, the lectin derived carbohydrate binding peptide should notbe toxic, and should be sufficiently soluble at the required dosages,which will typically range from about 0.5-50 mg/kg of body weight. Inthis regard, it is art recognized that the solubility of lectin derivedcarbohydrate binding peptides can be enhanced by attaching hydrophilicamino acid groups and can be reduced by attaching hydrophobic amino acidgroups from the carboxyl terminal and/or amino terminal positions of thepeptide.

The invention further contemplates fragments or derivatives of peptidescapable of binding terminally linked α-sialic acid(2→6)βGal- and/orα-sialic acid(2→3)βGal- structures which peptides have been modified torender them non-toxic e.g., by chemical derivatization, mutagenesis,etc., while still retaining the ability to bind such terminally linkedstructures.

The subject invention provides, in particular, methods for suppressingcell-mediated immune responses in mammals including cell-mediatedinflammatory responses or disorders by the administration of aneffective amount of one or more lectin derived carbohydrate bindingpeptides capable of binding terminally linked α-sialic acid(2→6)βGal-and/or α-sialic acid(2→3)βGal- structures or molecules/cell surfacescomprising such structures.

The cell-mediated immune responses or disorders treatable by the subjectinvention include inflammatory immune reactions involving specific andnon-specific defense systems. As discussed above, such conditionsinclude antibody responses to antigens, such as viruses, allergens,delayed-type hypersensitivity, autoimmune disorders such as rheumatoidarthritis and lupus, post-ischemic leukocyte mediated tissue damage(reperfusion injury), frost-bite injury or shock-acuteleukocyte-mediated lung injury (e.g., acute respiratory distresssyndrome), asthma, traumatic shock, septic shock, nephritis, and acuteand chronic inflammation, including atopic dermatitis, psoriasis, andinflammatory bowel disease. Further, inflammatory disorders treatable bythe subject invention may include platelet-mediated pathologies such asatherosclerosis and clotting disorders.

Inflammatory conditions of special interest include delayed typehypersensitivity reactions, reperfusion, and acute leukocyte-mediatedlung injury (ARDS).

This invention provides a generic method by which cell-mediated immuneresponses such as cell-mediated inflammatory responses or disorders inmammals (e.g., humans) may be suppressed by the administration of aneffective amount of one or more lectin derived carbohydrate bindingpeptides or fragments or derivatives thereof capable of bindingterminally linked α-sialic acid(2→6)βGal- and/or α-sialic acid(2→3)βGal-structures or molecules/cell surfaces comprising such structures. In apreferred embodiment, the invention provides methods by whichinflammatory responses or disorders may be treated or suppressed by theadministration of an effective amount of one or more peptides selectedfrom the group of peptides set forth in FIG. 1.

The subject invention further provides a general method for inhibitingimmune responses and cell adhesion events in mammals by theadministration of an effective amount of one or more lectin derivedcarbohydrate binding peptides or fragments or derivatives thereofcapable of binding terminally linked α-sialic acid(2→6)βGal- and/orα-sialic acid(2→3)βGal- structures or molecules/cell surfaces comprisingsuch structures. Such immune responses include cell mediated and humoralimmune responses. As has been discussed, such immune responses include,in particular, inflammatory responses or inflammatory disorders.

The invention further provides methods for affecting the induction ofimmune responses to antigens comprising administering to a mammal anantigen in conjunction with one or more lectin derived carbohydratebinding peptides capable of binding terminally linked α-sialicacid(2→6)βGal- and/or α-sialic acid(2→3)βGal- structures ormolecules/cell surfaces comprising such structures. For example,administration of an effective amount of the SPYGRC (amino acids 18-23of SEQ ID NO:3) hexapeptide to a mammal with an antigen will modulatethe induction of the immune response in the mammal to the antigen.Accordingly, the subject lectin derived carbohydrate binding peptidesmay comprise applicability as immune modulators, which may beadministered in conjunction with vaccines, artificial organs or tissuetransplants, and allogeneic organ and tissue transplants as a means formodulating the immune response to foreign antigens comprised therein.

It has further been found that the subject lectin derived carbohydratebinding peptides capable of binding terminally linked α-sialicacid(2→6)βGal- and/or α-sialic acid(2→3)βGal- structures ormolecules/cell surfaces comprising such structures, when administered inan effective amount to a mammal which has been immunized with aparticular antigen, result in the induction of long term tolerance tosaid antigen. In this regard, administration is conducted after onset ofthe secondary immune response but at or prior to one-half the periodrequired for maximal inflammation.

In particular, it has been found that administration, during thecritical period set forth above, of an effective amount of the S3P9a(SEQ ID NO:9), the ACS2P1 (2275), (amino acids 9-23 of SEQ ID NO:3) andthe SPYGRC (2283) (amino acids 18-23 of SEQ ID NO:3) peptidesillustrated in FIG. 1 to mammals that have been immunized with anantigen, results in said mammals exhibiting a reduced immune responseupon subsequent challenge(s) with said antigen (FIG. 3). Thus, thesubject lectin derived carbohydrate binding peptides have applicabilityas tolerogens. Given this property, such lectin derived carbohydratebinding peptides or fragments or derivatives thereof may be especiallysuitable for use in the treatment of allergic disorders sinceadministration of "tolerogenic" derivatized allergens is a known meansfor treating allergic disorders.

The subject invention further provides methods for inhibiting theadhesion of certain cell types e.g., tumor cells and polymorphonuclearcells (PMN's)! to endothelial cells. In this regard, the art suggeststhat tumor metastasis involves tumor cell adhesion to selectin bearingcells. In this regard, circulating cancer cells apparently takeadvantage of the body's normal inflammatory mechanisms and bind to areasof blood vessel walls were the endothelium is activated and,accordingly, contains selectins. As noted previously, the putativereceptors for such selectins contain terminally linked α-sialicacid(2→6)βGal- and/or α-sialic acid(2→3)βGal-structures. Therefore,administration of lectin derived carbohydrate binding peptides capableof binding terminally linked α-sialic acid(2→6)βGal- and/or α-sialicacid(2→3)βGal- structures should provide a method for inhibitingmetastasis. For example, the subject lectin derived carbohydrate bindingpeptides or fragments or derivatives thereof may be administered before,during or after cancer surgery or biopsy as a means for inhibitingmetastasis of tumor cells which may be released into the circulatorysystem during surgery. In these methods, the subject lectin derivedcarbohydrate binding peptides are administered either prior to, at thetime of surgery or biopsy, or shortly thereafter. Prior administrationis typically no more than about 5 hours prior to surgery or biopsy andadministration and subsequent administration is typically no more thanabout 15 hours after surgery or biopsy. In either case, administrationis either continuous or intermittent but preferably continuous.

In the methods pertaining to suppression of cell-mediated inflammatoryreactions or disorders arising from injury or antigen exposure, thesubject lectin derived carbohydrate binding peptides or fragments orderivatives thereof are administered after initiation of the mammalsimmune response but at or prior to one-half the period required formaximum inflammation to the antigen exposure or injury. Preferably, thesubject lectin derived carbohydrate binding peptides are administeredabout 1-10 hours after initiation of the immune response, and morepreferably about 1-5 hours after initiation of the immune response.However, the specific time for administration will vary dependent uponthe particular antigen/injury and the lectin derived carbohydratebinding peptide which is administered.

In the methods pertaining to modulating the induction of an immuneresponse to an antigen in a non-sensitized mammal, an effective amountof the subject lectin derived carbohydrate binding peptides orderivatives thereof will be administered in conjunction with theantigen. Typically, such conjunctive administration is simultaneous withantigen administration but can be up to ±3 hours from the time ofantigen administration.

In the methods pertaining to induction of long term tolerance to anantigen, an effective amount of the subject lectin derived carbohydratebinding peptides or fragments or derivatives thereof will generally beadministered after antigen challenge to a sensitized mammal. Inparticular, administration is after initiation of the mammal's secondaryimmune response to the antigen challenge but at or prior to one-half theperiod required for maximum inflammation to the antigen challenge.Preferably, the subject lectin derived carbohydrate binding peptides areadministered about 1-10 hours after initiation of the immune response tothe antigen challenge, and more preferably about 1-5 hours afterinitiation of the immune response to the antigen challenge. However, thespecific time for administration will vary dependent upon the particularantigen and the lectin derived carbohydrate binding peptide which isadministered.

Generally, the subject lectin derived carbohydrate binding peptides orderivatives thereof will be administered parenterally, e.g., byintramuscular or intravenous routes. However, other dosage forms shouldalso be suitable including, e.g., oral, transdermal, rectal,intratracheal, and intranasal formulations. For example, intranasal andintratracheal formulations may be preferred if the inflammatorycondition treated involves lung inflammation, e.g., acute respiratorydistress syndrome (ARDS). In contrast, an oral formulation would likelybe preferred if the inflammatory condition treated involves thedigestive tract, e.g., inflammatory bowel disease.

Pharmaceutical compositions for use in the subject invention willgenerally comprise an effective amount of one or more of the subjectpeptides or derivatives thereof capable of binding terminally linkedα-sialic acid(2→6)βGal- and/or α-sialic acid(2→3)βGal- structures incombination with a pharmaceutically acceptable carrier and/orexcipients. The particular pharmaceutically acceptable carrier andexcipients will vary dependent upon the dosage form. In one embodiment,several of the subject peptides or derivatives thereof are mixed intothe pharmaceutical composition to form a "cocktail" having enhancedactivity.

Parenteral dosage forms may contain phosphate buffered saline as acarrier, while intranasal formulations will comprise inhalants, and oraldosage forms may comprise enteric coatings. The selection of suitablecarriers and excipients and formulation of different dosage forms iswell within the level of ordinary skill in the pharmaceutical art.

As noted above, the subject lectin derived carbohydrate binding peptidesor derivatives thereof are administered in effective amounts. Aneffective amount is an amount sufficient to obtain the desired therapywithout causing undue toxicity to the mammal. Preferably, the subjectpeptides are administered at dosages ranging from about 0.5 to 50 mg/kgbody weight, with 5-10 mg/kg being most preferred for each of the abovecited methods. The specific dose employed is regulated by the particularcell-mediated immune response being treated as well as by the judgementof the attending clinician depending upon factors such as the severityof the adverse immune response, the age and general condition of thepatient, and the like.

Generally, the methods of the present invention will involveadministration of a single dose of the subject lectin derivedcarbohydrate binding peptides. However, the invention furthercontemplates repeated administration of the subject lectin derivedcarbohydrate binding peptides or derivatives thereof. Repeatedadministration of these peptides may be desirable, e.g., in thetreatment of chronic or sustained inflammatory disorders, such as,rheumatoid arthritis, acute and chronic inflammation, psoriasis,inflammatory bowel disorders, and autoimmune disorders associated withinflammatory responses, such as lupus, multiple sclerosis or rheumatoidarthritis.

It is also contemplated that the subject peptides and derivatives areuseful as receptor-targeted antibacterial and anti-viral drugs whereinthe bacteria, virus, or toxin produced therefrom employs a terminallylinked α-sialic acid(2→6)βGal- and/or α-sialic acid(2→3)βGal- structureas the receptor site on a cell of the targeted mammalian host.

Such bacteria/virus and/or toxins include, by way of example, influenzavirus, pertussis toxin, cholera toxin, and the like. Such methods areillustrated in the examples hereinbelow wherein in vitro assaysdemonstrate the ability of two of the subject lectin derivedcarbohydrate binding peptides to neutralize the effects of pertussistoxin on Chinese Hamster Ovary cells.

Accordingly, when administered in effective amounts, the subject lectinderived carbohydrate binding peptides are useful in methods forinhibiting inoculation in mammalian hosts of bacterial/viral agentsand/or their toxins which employ a terminally linked α-sialicacid(2→6)βGal- and/or α-sialic acid(2→3)βGal- structure as the receptorsite on a cell of the targeted mammalian host thereby inhibiting thelikelihood that the mammalian host will become afflicted with thedisease produced by the bacterial/viral agent and/or its toxin.

Effective amounts of the subject lectin derived carbohydrate bindingpeptides or derivatives thereof will preferably be dosages ranging fromabout 0.5 to 50 mg/kg body weight, with 5-10 mg/kg being most preferred.

3. EXAMPLES

In order to fully illustrate the present invention and the advantagesthereof, the following specific examples are given, it being understoodthat these examples are intended to be illustrative only and in nowiselimitative of the scope of the present invention.

In these examples as well as in the application, all sugars disclosedherein are in their D form except for fucose which is in its L form andall amino acids are conventional.

In these examples, unless otherwise defined below, the abbreviationsemployed herein have their generally accepted meaning:

    ______________________________________                                        ABTS =       2,2'-azino-bis(3-ethylbenzathiazoline-6-                                      sulfonic acid)                                                   BSA =        Bovine serum albumin                                             cm =         centimeter                                                       HPLC =       High Pressure Liquid Chromatography                              MAL =        Maackia amurensis                                                mg =         milligram                                                        mM =         millimolar                                                       mm =         millimeter                                                       ng =         nanogram                                                         nm =         nanometer                                                        PBS =        phosphate buffered saline                                        PT =         Pertussis toxin                                                  SNA =        Sambucus nigra                                                   μg =      microgram                                                        μl =      microliter                                                       μM =      micromolar                                                       μmol =    micromole                                                        v/v =        volume/volume                                                    ______________________________________                                    

Unless otherwise indicated, all temperatures are in degrees Celsius(°C.). Also, as noted previously, all amino acid residues recited hereinemploy their conventional one letter abbreviation.

General Procedures

All of the reagents used in Examples 1-10 were obtained from SigmaChemical Company, St. Louis, Mo., except for pertussis toxin (PT) whichwas obtained from Connaught Center for Biotechnology Research,Willowdale, Ontario, Canada; SNA-, WGA- and MAL-biotin, which wereobtained from Boehringer Mannheim, Dorval, Quebec; and IODO-GEN whichwas from Pierce Chemical Co., St. Louis, Mo. The acetylated andbiotinylated analogs of the peptide S2P1 (amino acids 9-23 of SEQ IDNO:3) were prepared using conventional methods. PT-biotin as well asasialo- and asialoagalactofetuin were prepared as describedearlier³⁰,44. Removable flat-bottomed microtiter well strips ofImmunulon 2 were from Dynatech, Alexandria, Va.

EXAMPLE 1

Synthesis of synthetic peptides

Peptides corresponding to amino acid sequences found in the S2 and S3subunits of PT were synthesized using an ABI 403A peptide synthesizer(Applied Biosystems, Inc., Foster City, Calif.), then cleaved from theresin by HF and purified by reversed-phase HPLC on a Vydac C4semipreparative column. All synthetic peptides used in ELISA inhibitionassays were >95% pure as judged by analytical HPLC, and their amino acidanalyses were in good agreement with the theoretical compositions.

Regions in the S2 and S3 subunits of PT that correspond to the variableamino acid sequences distinguishing the S2 from the S3 subunit weresecured as above. These peptide sequences were chosen, in part, based ontheir high index of hydrophilic β-turns as judged by secondary structureprediction analysis⁶⁰,61. Upon careful examination of the PT sequences,20 peptides were synthesized including those containing amino acidresidues 62-73 of WGA (SEQ ID NO:11). The acetylated version of S2P1(amino acids 9-23 of SEQ ID NO:3) was also prepared in order to bettermimic the native peptide backbone. The synthesized peptides are setforth in Table I below:

                                      TABLE I                                     __________________________________________________________________________    Peptide  Position                                                                           Sequence*              SEQ ID NO                                __________________________________________________________________________     1. S2P1  9-23                                                                              PQEQITQHGSPYGRC         3 (aa 9-23)                              2. ACS2P1-b                                                                            9-23                                                                              BIOTIN-PQEQITQHGSPYGRC-CO--NH.sub.2                                                                   3 (aa 9-23)                              3. S2 (14-23)                                                                         14-23                                                                              TQHGSPYGRC              3 (aa 14-23)                             4. S2-b 14-23                                                                              BIOTIN-TQHGSPYGRC       3 (aa 14-23)                             5. S2P2  1-23                                                                              STPGTVIPPQEQITQHGSPYGRC                                                                               3                                        6. SPYGRC                                                                             18-23                                                                              SPYGRC-CO--NH.sub.2     3 (aa 18-23)                             7. SPYGRC-b                                                                           18-23                                                                              BIOTIN-SPYGRC-CO--NH.sub.2                                                                            3 (aa 18-23)                             8. S2P3 78-98                                                                              GAFDLKTTFCIMTTRNTGQPA   4                                        9. S2P4 138-154                                                                            YDGKYWSMYSRLRKMLY       5 (aa 16-32)                            10. S2P6 123-154                                                                            FVRSGQPVTGACTSPYDGKYWSMYSRLRKMLY                                                                      5                                       11. S3P1  9-23                                                                              PKALFTQQGGAYGRC         6 (aa 9-23)                             12. S3P2  1-23                                                                              VAPGlVIPPKALFTQQGGAYGRC                                                                               6                                       13. S3P3  87-108                                                                            CITTIYKTGQPAADHYYSKVTA  7 (aa 10-31)                            14. S3P4  78-108                                                                            AGFIYRETFCITTIYKTGQPAADHYYSKVTA                                                                       7                                       15. S3P5 134-154                                                                            CASPYEGRYRDMYDALRRLLY   8                                       16. S3P9a                                                                              110-127                                                                            RLLASTNSRLCAVFVRDG      9                                       17. S2(WGA)#                                                                           --   PQEQITQHGSQYGYC        10                                       18. S2(WGA)-b                                                                          --   BIOTIN-PQEQITQHGSQYGYC 10                                       19. WGA(62-73)                                                                         --   SQYGYCGFGAEY           11                                       20. WGA(62-73)-b                                                                       --   BIOTIN-SQYGYCGFGAEY    11                                       __________________________________________________________________________     *Underlined sequences correspond to sequences found in the PT peptide tha     showed homology to the sequence SQYGHC (SEQ ID NO: 12) found in the           binding site of WGA isolectin 2 (24).                                         #PT S2P1 sequence (amino acids 9-23 of SEQ ID NO: 3) inserted with the WG     hexapeptide sequence                                                          (SQYGYC) (amino acids 10-15 of SEQ ID NO: 10)                                 aa amino acid                                                                 b biotin                                                                 

The above peptides were tested for their ability to bind to terminallylinked α-sialic acid(2→6)βGal- and α-sialic acid(2→3)βGal- structures inthe examples below. In these examples, the peptides were first screenedfor their ability to inhibit binding of different lectins (PT, SNA, andMAL) known to bind to fetuin, a carbohydrate containing multiple copiesof the αNeu5Ac(2→3)βGal(1→4)βGlc- structure.

EXAMPLE 2

Binding Inhibition Assays (Initial Peptide Screening)

Microtiter wells were coated with 100 μl of fetuin or asialofetuin (50μg/ml) in 50 mM sodium phosphate buffer (pH 6.8) containing 5 mM MgCl₂and 15 mM NaN₃ for 16 hours at 4° C. The solution was removed byaspiration and replaced with 100 μl of 1% BSA in PBS containing 0.05%Tween 20 (PBST). After incubation for 2-4 hours at room temperature, themicrotiter wells were washed four times with 300 μl of PBST. Peptidesranging in concentration from 0.5 to 4.5 mg/ml in PBS (40 μl ) wereadded to each well and PT-biotin (10 μl containing 10 ng in PBS) wasthen added to the microtiter wells. After incubating for 1 hour, thebinding reaction was stopped by aspirating the solutions and the platewas washed with PBST (300 μl). Horseradish peroxidase-conjugated avidin(100 μl 1/3000 dilution in PBST to a concentration of 0.3 μg/ml) wasthen added to the wells, and the plates were incubated at ambienttemperature for 1 hour. After washing the wells as described above, thesubstrate solution 1 mM ABTS in 5 mM citrate buffer, pH 4.2, containing0.1% hydrogen peroxide, v/v) was added and the plates were incubated for30 minutes. Color development was recorded at 405 nm using a TitertekMultiskan MC plate reader. Maximum binding was determined in the absenceof peptide, and background binding was measured in wells coated with BSAonly. Binding assays for each peptide were done in duplicate. Bindinginhibition experiments utilizing SNA-, WGA-, and MAL-biotin wereperformed as described above using 10 ng of each biotinylated lectin inPBS.

The panel of 20 peptides were assayed for the ability to inhibit PT-,WGA-, MAL-, and SNA-biotin binding to fetuin or asialofetuin. Thebiotinylated plant lectins have proved to be useful controls, since wehave previously showed that these lectins possess similar bindingspecificities as PT²⁵,46. The results from this example are set forth inTable 2 below. These results indicate that peptides S2P3 (SEQ ID NO:4)and S2P6 (SEQ ID NO:5) inhibited PT biotin binding by 15-20% both tofetuin as well as asialofetuin. Two additional peptides derived from theS3 subunit (S3P3 (amino acids 10-31 of SEQ ID NO:7) and S3P5 (SEQ IDNO:8)) were found to reduce binding of PT to asialofetuin only. Themajority of the other peptides displayed in Table 2 exhibited eithermarginal inhibition or enhancement in binding of biotinylated PT tofetuin or asialofetuin. Peptides S2P1 (amino acids 9-23 of SEQ ID NO:3),ACS2P1 (amino acids 9-23 of SEQ ID NO:3) and S2P2 (SEQ ID NO:3) showednearly a 2-fold enhancement of PT-biotin binding relative to controlexperiments done in the absence of peptide.

Peptide S2P3 (SEQ ID NO:4) was found to inhibit binding to fetuin of allthe biotinylated lectins. However, it is noted that peptides S2P1 (aminoacids 9-23 of SEQ ID NO:3) and S2P2 (SEQ ID NO:3), which showedenhancement of binding of PT to fetuin, inhibited WGA binding activity.

                                      TABLE II                                    __________________________________________________________________________    Percent Changes in Binding Activity                                                        Concentration                                                                        PT/  MAL/ SNA/                                                                              WGA/                                                                              PT/                                     Peptide                                                                             SEQ ID NO                                                                            (mg/mL)                                                                              fetuin*                                                                            fetuin                                                                             fetuin                                                                            fetuin                                                                            asialo-fetuin                           __________________________________________________________________________    S2P1   3 (aa 9-23)                                                                         1.2     +85 ± 4                                                                         +8 ± 4                                                                          -5 ± 1                                                                        -18 ± 2                                                                        +113(1)                                 ACS2P1                                                                               3 (aa 9-23)                                                                         1.0     +89 ± 3                                                                        +16 ± 9                                                                          -3 ± 1                                                                        -22 ± 6                                                                        +190 ± 23                            S2 (14-23)                                                                           3 (aa 14-23)                                                                        1.0     +68 ± 9                                                                        +32 ± 1                                                                          +1 ± 2                                                                         +7 ± 1                                                                        ND                                      S2P2   3     1.1     +58 ± 4                                                                        +14 ± 2                                                                          -1 ± 1                                                                        -15 ± 2                                                                         +33(1)                                 S2P3   4     0.51    -21 ± 0                                                                        -18 ± 2                                                                         -15 ± 4                                                                        -56 ± 8                                                                         -15(1)                                 S2P4   5 (aa 16-32)                                                                        0.98    +14 ± 22                                                                        +2(1)                                                                              +3(1)                                                                            +15 ± 1                                                                         -3(1)                                  S2P6   5     0.85    -28 ± 6                                                                        +18 ± 16                                                                         +2 ± 2                                                                        +57 ± 0                                                                         -18(1)                                 S3P1   6 (aa 9-23)                                                                         3.2     +54 ± 25                                                                        +4 ± 6                                                                          -2 ± 1                                                                         -9 ± 4                                                                         +45 ± 16                            S3P2   6     3.1     +77 ± 59                                                                       +12 ± 16                                                                         +3 ± 3                                                                         +9 ± 4                                                                         +82 ± 11                            S3P3   7 (aa 10-31)                                                                        4.5     +19 ± 67                                                                       +13 ± 15                                                                         +3 ± 0                                                                         +6 ± 3                                                                         -28 ± 7                             S3P4   7     3.3     +12 ± 2                                                                         +7 ± 1                                                                          +1(1)                                                                            +13 ± 1                                                                         +75 ± 50                            S3P5   8     0.48    +14 ± 6                                                                         +3 ± 6                                                                          0(1)                                                                             +35 ± 8                                                                         -26 ± 10                            S3P9a  9     1.7    +328 ± 10                                                                       +18 ± 6                                                                         -41 ± 3                                                                        ND  ND                                      SPYGRC                                                                               3 (aa 18-23)                                                                        4.4     +34 ± 2                                                                        -16 ± 0                                                                          -2 ± 2                                                                         +7 ± 3                                                                         +7 ± 9                              S2(WGA)                                                                             10     1.0     +79 ± 15                                                                       +22 ± 3                                                                          0 ± 1                                                                          -4 ± 3                                                                        ND                                      WGA   11     1.0     -32 ± 5                                                                         -1 ± 4                                                                          -3 ± 2                                                                         -9 ± 1                                                                        ND                                      (62-73)                                                                       __________________________________________________________________________     *Biotinylated lectin/glycoprotein bound to microtiter wells. Positive         signs preceding the numbers in this table indicate enhancement and            negative signs indicate inhibition of binding.                                (1) One determination only.                                                   aa amino acid                                                                 ND Not determined.                                                       

EXAMPLE 3

Binding Inhibition Assays (Determination of IC 50 Values)

Peptides found to have inhibitory activity in the initial screeningexperiments were further analyzed in binding inhibition experiments todetermine the concentration of peptide that was required to reducebinding by 50% (IC₅₀ values).

Inhibition experiments were performed by a method similar to thatdescribed in Example 2 with 2-fold dilutions of PT peptide in PBS,except that microtiter wells were coated with 3 μg/ml fetuin orasialofetuin. Binding assays for each inhibitor concentration were doneat least in duplicate, and the average value varied less than 15%. Theconcentration of peptide required for 50% inhibition (IC₅₀) wasdetermined by plotting the amount of binding observed in the presence ofpeptide inhibitor as a percent of the maximum binding achieved withoutinhibitor.

Two of the peptides derived from the S2 subunit were able to inhibitPT-biotin binding to fetuin at submillimolar concentrations, but theywere unable to inhibit PT-biotin binding to asialofetuin in subsequentexperiments (Table III). The two peptide sequences from the S3 subunit(S3P3 (amino acids 10-31 of SEQ ID NO:7) and S3P5 (SEQ ID NO:8)) werefound to inhibit PT-biotin's interaction with asialofetuin in aconcentration-dependent manner, but their IC₅₀ values are above thesolubility limits of the peptide in PBS. The peptide S2P3 (SEQ ID NO:4)was also very active at inhibiting the interaction of MAL- andWGA-biotin with fetuin. Upon closer examination this peptide proved tobe non-inhibitory for SNA-biotin. Two additional peptides from S2 (S2P1(amino acids 9-23 of SEQ ID NO:3) and S2P2 (SEQ ID NO3)) were active atinhibiting WGA-biotin binding at millimolar concentrations, suggestingthat these peptide sequences may also be important for interacting witholigosaccharide in the binding site of WGA.

                  TABLE III                                                       ______________________________________                                        Concentrations of PT Peptides and WGA Peptides Resulting                      in 50% Inhibition of Biotinylated PT and Lectins Binding                      to Fetuin or Asialofetuin                                                     Biotinylated Lectin                                                           (glycoprotein)                                                                             Peptide    SEQ ID NO  IC.sub.50 (mM)                             ______________________________________                                        PT-b (fetuin)                                                                              S2P3        4         0.19 ± 0.09                             PT-b (fetuin)                                                                              S2P6        5         0.22 ± 0.06                             WGA-b (fetuin)                                                                             ACS2P1      3 (aa 9-23)                                                                             3.25 ± 0.45*                            WGA-b (fetuin)                                                                             S2P2        3         SI                                         WGA-b (fetuin)                                                                             S2P3        4         0.14 ± 0.03                             MAL-b (fetuin)                                                                             S2P3        4         0.86 ± 0.02                             PT-b (asialofetuin)                                                                        S3P3        7 (aa 10-31)                                                                            SI                                         PT-b (asialofetuin)                                                                        S3P5        8         SI                                         SNA-b (fetuin)                                                                             S3P9a       9         2.74 ± 0.86                             PT-b (fetuin)                                                                              WGA (62-73)                                                                              11          1.5 ± 0                                WGA-b (fetuin)                                                                             WGA (62-73)                                                                              11          3.4 ± 0                                ______________________________________                                         SI Slightly inhibitory, but the concentration of peptide required for 50%     inhibition of binding is above the solubility limit of the peptide.           *Unacetylated form of S2P1 (amino acids 9-23 of SEQ ID NO: 3) inhibited t     the same extent as the acetylated form.                                       aa amino acid                                                            

Since three of the peptides derived from the S2 subunit of PT were ableto inhibit WGA binding, the amino acid sequences which constitute thesialic acid binding site of WGA were closely examined to determine ifthere were any homologies with the inhibitory PT S2 peptide sequences.One short 6-amino acid sequence (SQYGHC) (SEQ ID NO:12) corresponding toamino acids 62-67 in WGA isolectin 2 displayed reasonable homology witha sequence found both in the S2P1 (amino acids 9-23 of SEQ ID NO:3) andS2P2 (SEQ ID NO:3) peptides (SPYGRC, amino acids 18-23 of SEQ ID NO:3)from PT. This short sequence in WGA is responsible for binding with thecarbonyl of the N-acetyl group of sialic acid or N-acetyl-glucosamine(i.e. serine 62) through a hydrogen bond. Non-polar interactions betweenthe aromatic side chains of tyrosine 64 as well as histidine 66 interactwith the glycerol side chain of sialic acid and the pyranose ring ofsialic acid or N-acetyl-glucosamine, respectively⁶². The otherinhibitory peptide, S2P3 (SEQ ID NO:4), did not display any goodhomology with sequences responsible for interacting with sialic acid inWGA. This indicates that other motifs may also be functionally importantfor interaction with sialic acid.

EXAMPLE 4

Binding Inhibition Studies Utilizing Biotinylated and Acetylated S2P1(amino acids 9-23 of SEQ ID NO:3)

From the initial peptide screening results (Table II), peptides S2P1(amino acids 9-23 of SEQ ID NO:3) and S2P2 (SEQ ID NO:3) showed a 2-foldenhancement in binding of PT-biotin to fetuin relative to controlexperiments. One possible explanation for the enhancement may be theability of the peptide to form a bridge between PT-biotin and fetuin. Inorder for the peptide to act as a bridging molecule, the peptide mustcontain both a recognition site for fetuin as well as a sequence whichbinds to PT itself. To answer this question an acetylated andbiotinylated form of the peptide S2P1 (amino acids 9-23 of SEQ ID NO:3)(biotinylated at the terminal proline) was prepared to determine if wecould measure direct binding of the peptide to PT and fetuin.

Binding assays were done as described in Example 2 in PBS by usingACS2P1-biotin (amino acids 9-23 of SEQ ID NO:3) at a concentration of 10μg/ml in PBS. Assays were carried out for 1 hour at room temperature andthe amount of biotinylated peptide bound to fetuin, determined by usingavidin-peroxidase.

From these direct binding studies, a concentration-dependent bindingboth to fetuin as well as to PT immobilized in microtiter wells wasobserved. Furthermore, the binding of ACS2P1-biotin (amino acids 9-23 ofSEQ ID NO:3) to PT could be inhibited by fetuin (IC₅₀ =50 μM; n=2)indicating that the biotinylated peptide bound at or adjacent to thefetuin binding site in PT.

In view of the above, it was concluded that because of potentialbridging by the peptide between the lectin and the fetuin, any of thepeptide sequences set forth in Table I which caused reduction in thebinding of any of the lectins to fetuin were able to bind to terminallylinked α-sialic acid(2→6)βGal- and α-sialic acid(2→3)βGal- structures.

EXAMPLE 5

Binding Inhibition Assays Using Oligosaccharides to Inhibit the Bindingof ACS2P1-biotin (amino acids 9-23 of SEQ ID NO:3)

The binding results obtained for ACS2P1-biotin (amino acids 9-23 of SEQID NO:3) described in Example 4 above indicate a direct involvement ofcarbohydrate moiety with the lectin through a biotinylated peptidebridge.

In order to assess the binding to α-sialic acid(2→6)βGal- and α-sialicacid(2→3)βGal- structures by this peptide as well as the hexapeptideSPYGRC (amino acids 18-23 of SEQ ID NO:3) which only exhibited reductionin the binding of one lectin to fetuin (Table II), inhibitionexperiments with simple saccharides were conducted to determine if theinteraction was carbohydrate-dependent. The simple sugars chosen weresialic acid, sialyllactose from bovine colostrum (contains a mixture ofα(2→6) and α(2→3) linked sialic acid structures), lactose, andN-acetylglucosamine.

Binding inhibition assays were done using sialic acid, sialyllactose,lactose, or N-acetylglucosamine at a concentration of 8 mM in PBS.Inhibition assays were done for 1 hour as described above (e.g., Example2) at room temperature and the amount of biotinylated peptide bound tofetuin (3μg/ml) quantitated by using avidin-peroxidase. The pH of freesialic acid was carefully monitored and adjusted to physiological pHvalues with dilute sodium hydroxide. The results of this example are setforth in Table IV below:

                  TABLE IV                                                        ______________________________________                                        Inhibition of Binding of ACS2P1-biotin (amino acids 9-23 of SEQ ID            NO: 3) and SPYGRC-biotin (amino acids 18-23 of SEQ ID NO: 3) to               Fetuin by Simple Saccharides*                                                 Percent Changes in Binding Activity                                                   ACS2P1-biotin   SPYGRC-biotin                                                 (amino acids 9-23 of                                                                          (amino acids 18-23 of                                         SEQ ID NO: 3)   SEQ ID NO. 3)                                         Saccharide                                                                            (n = 3)         (n = 3)                                               ______________________________________                                        Sialic acid                                                                           -19 ± 6       +9 ± 8                                            Sialyllactose                                                                         -46 ± 3      -24 ± 6                                            Lactose +74 ± 7      +20 ± 11                                           N-Acetylglu-                                                                          -10 ± 2       +6 ± 5                                            cosamine                                                                      Sucrose  0 ± 3       ND                                                    ______________________________________                                         *Inhibition experiments were carried out as outlined above using 0.1 μ     or 0.5 μg of ASCS2P1biotin (amino acids 9-23 of SEQ ID NO: 3) and          SPYGRCbiotin (amino acids 18-23 of SEQ ID NO: 3) respectively. Positive       signs preceding the number in this table indicate enhancement and negativ     signs indicate inhibition of binding to fetuin.                               ND Not determined                                                        

The above results indicate that both peptides tested were able to bindto terminally linked α-sialic acid(2→6)βGal- and α-sialic acid(2→3)βGal-structures. Control experiments indicated that sialic acid was necessaryfor high affinity interaction since lactose failed to reduce the bindingof ACS2P1-biotin (amino acids 9-23 of SEQ ID NO:3). N-acetylglucosaminewas also found to marginally inhibit the binding of biotinylated peptide(10±2%, n=3) suggesting that the ACS2P1-biotin (amino acids 9-23 of SEQID NO:3) may have weak affinity for the N-acetylglucosamine consistentwith the finding described below, which shows enhanced binding of thepeptide to asialoagalactofetuin relative to asialofetuin. This is alsoin good agreement with previous results, which indicate PT's bindingspecificities to be similar to those of WGA⁴⁴,52.

In view of the above, it was concluded that any of the peptides whichinhibit the binding of fetuin to any of the lectins set forth in TableII above are effective in binding to terminally linked α-sialicacid(2→6)βGal- and α-sialic acid(2→3)βGal- structures. Such peptides areset forth in FIG. 1.

EXAMPLE 6

Screening of S2 and S3 Peptides from PT for the Ability to InhibitACS2P1-biotin (amino acids 9-23 of SEQ ID NO:3) Binding to Fetuin andAsialofetuin

Binding inhibition assays were conducted using S2 and S3 peptides fromPT in PBS as competitors for binding of ACS2P1-biotin (amino acids 9-23of SEQ ID NO:3) (10 μg/ml) to fetuin or asialofetuin. Inhibition assayswere done for 1 hour at room temperature, as described above, and theamount of biotinylated peptide bound to fetuin or asialofetuin (3 μg/ml)quantitated by using avidin-peroxidase.

The specificity and relative affinity of ACS2P1-biotin (amino acids 9-23of SEQ ID NO:3) for fetuin was determined by performing bindinginhibition experiments with the peptides shown in Table I. Bindinginhibition experiments (Table V) indicated that the ACS2P1 peptide(amino acids 9-23 of SEQ ID NO:3) bound to fetuin with high affinity(IC₅₀ =4.1 μM; n=2) when the unbiotinylated form of ACS2P1 (amino acids9-23 of SEQ ID NO:3) was used as competitor. The peptide S2P2, (SEQ IDNO:3) an extended version of S2P1 (amino acids 9-23 of SEQ ID NO:3) wasalso found to inhibit ACS2P1-biotin (amino acids 9-23 of SEQ ID NO:3)binding to fetuin but with a 10-fold decrease in affinity (IC₅₀ =42.5μM). Both peptides were unable to compete for the binding ofACS2P1-biotin (amino acids 9-23 of SEQ ID NO:3) to asialofetuin,indicating the importance of sialic acid for high affinity interactionwith the peptide. Similar hexapeptide sequences to those found withinthe S2P1 (amino acids 9-23 of SEQ ID NO:3) and S2P2 (SEQ ID NO:3)peptides are also present in a number of the peptides from Table I (seeunderlined segments). Each of these peptides were analyzed for theirability to inhibit ACS2P1-biotin (amino acids 9-23 of SEQ ID NO:3)binding to fetuin and asialofetuin at peptide concentrations 10-foldhigher than the IC₅₀ determined for the S2P1 (amino acids 9-23 of SEQ IDNO:3) peptide. None of the other peptides examined from Table I had theability to inhibit binding of ACS2P1-biotin (amino acids 9-23 of SEQ IDNO:3) to fetuin or asialofetuin to the same extent indicating that thepeptide sequence SPYGRC (amino acids 18-23 of SEQ ID NO:3) may play acrucial role for binding sialic acid. This is further confirmed by theinability of the peptides S3P1 (amino acids 9-23 of SEQ ID NO:6) andS3P2, (SEQ ID NO:6) which possess the strongest homologies to thesequence found in S2P1, (amino acids 9-23 of SEQ ID NO:3) to inhibitbinding even at high concentrations of peptide. The homologous sequencefound in the S3 peptides (GAYGRC) (amino acids 18-23 of SEQ IDNO:6)lacks the serine amino acid residue, which was shown to beimportant for forming an important hydrogen bond with the carbonyl ofthe N-acetyl group in the sialic acid binding site of WGA⁶². Thisindicates that the serine residue found in the S2P1 (amino acids 9-23 ofSEQ ID NO:3) peptide may function in an analogous manner in the bindingof S2P1 (amino acids 9-23 of SEQ ID NO:3)to sialic acid.

The results of this example are set forth in Table V below:

                  TABLE V                                                         ______________________________________                                        Screening of S2 and S3 Peptides from PT for the Ability                       to Inhibit ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3)                   Binding                                                                                         Concentration      Asialo-                                  Peptide                                                                              SEQ ID NO  (μM)    Fetuin.sup.1                                                                          fetuin.sup.1                             ______________________________________                                        ACS2P1 3 (aa 9-23)                                                                              4.1        -50       +101 ± 2                            S2P2   3          42.5       -50       +6   ± 1                            SPYGRC 3 (aa 18-23)                                                                             5520       -24  ± 2                                                                             -54  ± 3                            S2P3   4          22.4       +70  ± 11                                                                            +15  ± 11                           S2P4   5 (aa 16-32)                                                                             6.0        -8   ± 16                                                                            +2   ± 4                            S2P6   5          319        +49  ± 5                                                                             +18  ± 16                           S3P1   6 (aa 9-23)                                                                              1142       +245 ± 28                                                                            +4   ± 0                            S3P2   6          934        +88  ± 72                                                                            +12  ± 16                           S3P3   7 (aa 10-31)                                                                             16.1       -13  ± 16                                                                            -10  ± 11                           S3P4   7          6.4        +55  ± 26                                                                            +26  ± 2                            S3P5   8          22.5       -19  ± 4                                                                             -17  ± 8                            ______________________________________                                         The effect of S2 and S3 PT peptides on the binding of ACS2P1biotin (amino     acids 9-23 of SEQ ID NO: 3) (0.1 μg) to fetuin or asialofetuin.            Negative and positive values refert to percent inhibition or enhancement      respectively.                                                                 aa amino acid                                                                 .sup.1 Percent Change in Binding Activity                                

EXAMPLE 7

Iodination of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO:3)

Previous reports have suggested the importance of tyrosine amino acidresidues in the binding of PT to sialylated glycoprotein receptors⁴⁴,63.These reports were based on the observation that if PT is iodinated bythe conventional IODO-GEN procedure (selectively modifies tyrosineresidues) without first protecting the binding site for fetuin, thebinding activity of PT was reduced.

To determine if the tyrosine residue found in ACS2P1-biotin (amino acids9-23 of SEQ ID NO:3) plays a role in the binding activity, the peptidewas iodinated by the IODO-GEN procedure and its binding activitycompared with the uniodinated peptide. Specifically, ACS2P1-biotin(amino acids 9-23 of SEQ ID NO:3) (100 μg 0.052 μmol) in 100 μl of PBSwas placed in a 12×75-mm glass tube coated with IODO-GEN, and 0.1 mMsolution of NaI (50 μL 5 μmol) was added and gently mixed over a periodof 10 minutes at room temperature. The reaction was terminated byremoving the mixture from the IODO-GEN tube and the iodinated peptidewas purified on a Sephadex G-25 column (1×15 cm equilibrated with PBS).Individual fractions were analyzed for the presence of peptide bymeasuring absorbance at 220 nm, and the concentration was determined bycomparing the absorbance with underivatized ACS2P1-biotin (amino acids9-23 of SEQ ID NO:3). A sample of iodinated and underivatizedACS2P1-biotin (amino acids 9-23 of SEQ ID NO:3) were diluted to aconcentration of 10 μg/ml and analyzed for binding to fetuin coatedmicrotiter wells as described in Example 6. In addition, binding ofACS2P1-biotin (amino acids 9-23 of SEQ ID NO:3) (10 μg/ml) to fetuin,asialofetuin, and asialoagalactofetuin (each at 3 μg/ml) was done in asimilar manner.

The results of this experiment indicate that extensive iodination ofACS2P1-biotin (amino acids 9-23 of SEQ ID NO:3) reduced binding of thepeptide to fetuin by 58±3% (n=3), which suggests the importance of thetyrosine amino acid in addition to the serine for the binding activityof ACS2P1-biotin (amino acids 9-23 of SEQ ID NO:3) to terminally linkedα-sialic acid(2→6)βGal- and α-sialic acid(2→3)βGal- structures.

To determine whether the amino acid sequence in the S2 subunit of PTthat corresponds to the peptide S2P1 (amino acids 9-23 of SEQ ID NO:3)plays an actual role in the lectin-like binding activity of PT, thebinding specificity of the biotinylated peptide was compared with thatof PT using fetuin, asialofetuin, and asialoagalactofetuin. A previousreport had determined that the binding of ¹²⁵ I-PT to asialofetuin was53±7%, while asialoagalactofetuin was 81±8% relative to control bindingto fetuin⁴⁴. This is in good agreement with the results obtained withACS2P1-biotin (amino acids 9-23 of SEQ ID NO:3) which showed 45±9% and93±18% (n=3) binding to asialo- and asialoagalactofetuin, respectively,relative to fetuin. These results suggest that this amino acid sequencemay contain a portion of a lectin-like binding site in the S2 subunit ofPT, which is responsible for the binding results previously observed.

EXAMPLE 8

Binding assays were carried out essentially as described above usingmicrotiter wells that were coated with 50 μl of BSA glycoconjugate (50μg/ml) in 50 mM sodium phosphate buffer (pH 6.8) containing 5 mM MgCl₂and 15 mM NaN₃ for 16 hours at 4° C. The solution was removed byaspiration and replaced with 100 μl of 1% BSA in PBS containing 0.05%Tween 20 (PBST) and incubated at room temperature for an additional 2-3hours. The microtiter wells were washed four times with 300 μl of PBSTand then replaced with ACS2P1-biotin (amino acids 9-23 of SEQ ID NO:3)(0.1 μg) or SPYGRC-biotin (amino acids 8-23 of SEQ ID NO:3) (0.5 μg) in50 μl of PBS. After incubating for 1 hour, the binding reaction wasstopped by aspirating the solutions and the plate was washed with PBST(4×300 μl). Avidin-peroxidase (100 μl of a 1/3000 dilution of a 1 mg/mlsolution in PBST) was added and incubated for an additional 1 hour.After washing the wells as described above, the substrate solution (1 mMABTS in 5 mM citrate buffer, pH 4.2, containing 0.1% hydrogen peroxide,v/v) was added and the plates were incubated for 30 minutes. Bindingassays for each BSA conjugate were done in triplicate and backgroundbinding was measured in wells coated with BSA only. The extent ofbiotinylated peptide binding is expressed as a percentage relative tofetuin as shown in Table VI below:

                                      TABLE VI                                    __________________________________________________________________________    Binding of ACS2P1-biotin (amino acids 9-23 of SEQ ID NO: 3) and               SPYGRC-biotin (amino acids 18-23 of SEQ ID NO: 3) to BSA Conjugates*                            % Binding                                                                     Relative to      % Binding                                                    Fetuin of        Relative to Fetuin                                           ACS2P1-b (amino acids                                                                          of SPYGRC-b (amino acids                                                                         Common Name for                           9-23 of SEQ ID NO: 3)                                                                          18-23 of SEQ ID NO: 3)                                                                           Carbohydrate            Carbohydrate Structure of BSA Conjugate                                                         (n = 3)          (n = 3)            Structure               __________________________________________________________________________    αNeuAc(2-3)βGal(1-4)βGlcNAc-BSA                                                 117 ± 12      120 ± 7         SLacNAc                 αNeuAc(2-3)βGal(1-3)βGlcNAc-BSA                                                  70 ± 4        97 ± 12        SLe.sup.c               αNeuAc(2-6)βGal(1-4)βGlcNAc-BSA                                                  94 ± 2       122 ± 5         SLacNAc                 αNeuAc(2-3)βGal(1-4)βGlcNAc-BSA                                                  84 ± 3       120 ± 4         SLe.sup.x               (1-3)                                                                         αFuc                                                                    αNeuAc(2-3)βGal(1-3)βGlcNAc-BSA                                                  84 ± 14      103 ± 8         SLe.sup.a (C19.9)       (1-4)                                                                         αFuc                                                                    __________________________________________________________________________     *Experiments were done by coating 50 μg/mL BSAconjugate or fetuin and      probed with 0.1 μg ACS2P1biotin (amino acids 9-23 of SEQ ID NO: 3) or      0.5 μg of SPYGRYCb (amino acids 18-23 of SEQ ID NO: 3) for 1 hour at       room temperature.                                                        

EXAMPLE 9

Formation of a (amino acids 18-23 of SEQ ID NO:3) Conjugate

Streptavidin (50 μg) in PBS was combined with SPYGRC-biotin (amino acids18-23 of SEQ ID NO:3) (50 μl, 200 μg) and PBS (150 μl) and mixed for 1hour at room temperature. The reaction mixture was loaded onto aSephadex G-25 column (1×15 cm that had been equilibrated in PBS) andfractions collected. Protein containing fractions were analyzed by SDSPAGE gel electrophoresis and the molecular weight was determined. Theresults were consistent with the formation of a tetravalent complexbetween peptide and streptavidin. Lectin binding inhibition experimentswere carried out as described previously for determination of IC₅₀ 's.The results are set forth in Table VII below:

                                      TABLE VII                                   __________________________________________________________________________    The Effect of the Peptide SPYGRC or SPYGRC-biotin-Streptavidin (amino         acids 18-23 of SEQ ID NO: 3)                                                  Conjugate on the Binding of Biotinylated PT and Lectins to Fetuin*                     Concentration of                Concentration of                              SPYGRC Peptide (amino acids 18-23 of SEQ ID NO:                                                       % Changes in                                                                          Peptide-Streptavidin                                                                   % Changes in                Biotinylated lectin                                                                    (mg/ml)                 Binding Activity                                                                      Conjugate (μg/ml)                                                                   Binding                     __________________________________________________________________________                                                      Activity                    PT-b     4.4                     +34 ± 2                                                                            500      +13 ± 11                 MAL-b    4.4                     -16 ± 0                                                                            1.0      -50 ± 3                  SNA-b    4.4                      -2 ± 1                                                                            1.2      -50 ± 5                  WGA-b    4.4                      +7 ± 3                                                                            64       -50 ± 1                  __________________________________________________________________________     *Positive signs preceding the numbers in this table indicate enhancement      and negative signs indicate inhibition of binding.                       

EXAMPLE 10

Neutralization of Pertussis Toxin Binding to Chinese Hamster Ovary (CHO)Cell by Synthetic Peptides

Confluent monolayers of CHO cells were lifted from plastic tissueculture flasks with 0.25% trypsin and suspended at a concentration of5×10⁴ cells/ml in Ham's F12 media containing 10% fetal bovine serum(FBS). 100 μl of the cell suspension was added to 96 well tissue cultureplates and were allowed to establish contact with the plastic for 24 to48 hours. The expended media was then removed and filtered-sterilizedpeptide 5-fold dilutions ranging in concentration from 300 to 30femptogram per ml (80 μl) in Ham' F12 media containing FBS were added tothe CHO cells. 20 μl of a PT solution (final PT concentration 2.7 ng/ml)was then added to the tissue culture wells contain peptide. Theincubation mixtures were gently mixed and incubated at 37° C. for 1 hourin a CO₂ incubator. The incubation mixtures were then removed andreplaced with fresh media. The tissue culture plates were then incubatedfor 24 hours at 37° C., fixed with 100% methanol, and stained withGeimsa stain. Control experiments were done in the absence of peptidealone or in the absence of PT. All determinations were done intriplicate. Stained CHO cells were then examined for inhibition ofcharacteristic CHO cell clumping which is mediated by pertussis toxinbinding and scored as to whether there was greater than 50% inhibitionof clumping of CHO cells relative to control wells in the presence of PTonly. The results reported in Table VIII show the maximum peptideconcentration required to cause a 50% reduction in CHO cell clumping.

                  TABLE VIII                                                      ______________________________________                                        CHO Cell Neutralization Experiments Using PT S2 and S3 Peptides*                                    Maximal Inhibitory Concentration                        PT Peptide SEQ ID NO  for Greater than 50% Inhibition                         ______________________________________                                        ACS2P1     3 (aa 9-23)                                                                               96 pg/ml                                               SPYGRC     3 (aa 18-23)                                                                             480 pg/ml                                               S2P2       3          NI                                                      S3P1       6 (aa 9-23)                                                                              NI                                                      S3P2       6          NI                                                      S3P9a      9          NI                                                      ______________________________________                                         *CHO cell neutralization experiments were carried out using a PT              concentration of 2.4 ng/ml. CHO cell (5.4 × 10.sup.4 cells/ml) were     exposed to incubation mixtures containing peptide and PT for 1 hour at        room temperature. Control experiments were done in the absence of peptide     aa amino acid                                                                 NI No inhibitory at a peptide concentration of 300 ng/ml.                     pg picogram.                                                             

The above data demonstrates that at least certain of the subjectpeptides would be effective in inhibiting the attachment ofbacterial/viral agents and/or their toxins which utilize α-sialicacid(2→6)βGal- and/or α-sialic acid(2→3)βGal- structures as theattachment point on the surface of mammalian cells. Such agents/toxinsinclude known toxins such as pertussis toxin, cholera toxin, etc. and,accordingly, administration of an effective amount of at least one ofthe subject peptides to a mammal would be effective in inhibiting suchattachment.

Examples 11 and 12 below illustrate in vivo results for the subjectpeptides.

EXAMPLE 11

Inhibition of DTH Inflammatory Response

DTH inflammatory responses were measured using the mouse footpadswelling assay as described by Smith and Ziola⁶⁴. Briefly, groups ofBalb/c mice (about 19-20 grams each) were immunized with 100 μg of theOVA antigen containing 20μg of the adjuvant(DDA--dimethyldioctadecylammonium bromide) which also induces a stronginflammatory DTH response. Seven days later, each group of mice wasfootpad-challenged with 20 μg of the OVA antigen (without adjuvant). Theresulting inflammatory footpad swelling was measured with a MitutoyoEngineering micrometer 24 hours after challenge.

To assess the effect of different peptides on the inflammatory DTHresponse, groups of mice received 100 μg of the following peptides:ACS2P1 (2275) (amino acids 9-23 of SEQ ID NO:3), SPYGRC (2283) (aminoacids 18-23 of SEQ ID NO:3), and S3P9a (SEQ ID NO:9). These peptideswere injected as a solution into the tail vein, 5 hours after challenge.Control groups were left untreated or received 100 μL ofphosphate-buffered saline (PBS). The results of this experiment areshown in FIG. 2 which illustrates that the peptides employed wereeffective in reducing a DTH response in mice.

EXAMPLE 12

Persistence of Suppression of the DTH Inflammatory Response at 2 WeeksAfter Challenge

Identical groups of mice treated with the peptides in Example 11 above,were re-challenged with OVA antigen 2 weeks after primary immunization.Untreated controls responded with the usual degree of footpad swellingwhereas all other groups showed reduced footpad swelling. Specifically,these results are set forth in FIG. 3 which illustrates reduction in thedegree of footpad swelling in mice previously treated with the subjectpeptides.

In addition to providing suppression of antigen induced inflammation ina sensitized mouse, the above data demonstrate that treatment with thesubject peptides as per this invention also imparts tolerance to stilllater challenges from the same antigen.

In view of the fact that the immune system of mice serves as a goodmodel for the immune system of humans, the above data demonstrates thatthe subject peptides would be effective in suppressing cell-mediatedimmune responses in humans and, when the cell-mediated immune responseis to an antigen, this data also shows that the subject peptides wouldalso impart tolerance to later challenges to the human of that antigen.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 12                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note="X is selected from the                          group consisting of amino acids Tyr, Phe, Trp and                             His or peptide mimetics thereof"                                              (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note="X is selected from the                          group consisting of amino acids Tyr, Phe, Arg, Trp                            and His or peptide mimetics thereof"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       SerProXaaGlyXaaCys                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note="X is selected from the                          group consisting of amino acids Tyr, Phe, Trp and                             His of peptide mimetics thereof"                                              (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note="X is selected from the                          group consisting of amino acids Tyr, Phe, Arg,                                Trp, and His or peptide mimetics thereof"                                     (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note="4-6 amino acids"                                (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note="X is selected from the                          group consisting of amino acids Tyr, Phe, Trp and                             His or peptide mimetics thereof"                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       SerProXaaGlyXaaCysXaaXaa                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       SerThrProGlyIleValIleProProGlnGluGlnIleThrGlnHis                              151015                                                                        GlySerProTyrGlyArgCys                                                         20                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GlyAlaPheAspLeuLysThrThrPheCysIleMetThrThrArgAsn                              151015                                                                        ThrGlyGlnProAla                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       PheValArgSerGlyGlnProValIleGlyAlaCysThrSerProTyr                              151015                                                                        AspGlyLysTyrTrpSerMetTyrSerArgLeuArgLysMetLeuTyr                              202530                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ValAlaProGlyIleValIleProProLysAlaLeuPheThrGlnGln                              151015                                                                        GlyGlyAlaTyrGlyArgCys                                                         20                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AlaGlyPheIleTyrArgGluThrPheCysIleThrThrIleTyrLys                              151015                                                                        ThrGlyGlnProAlaAlaAspHisTyrTyrSerLysValThrAla                                 202530                                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CysAlaSerProTyrGluGlyArgTyrArgAspMetTyrAspAlaLeu                              151015                                                                        ArgArgLeuLeuTyr                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ArgLeuLeuAlaSerThrAsnSerArgLeuCysAlaValPheValArg                              151015                                                                        AspGly                                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      ProGlnGluGlnIleThrGlnHisGlySerGlnTyrGlyTyrCys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      SerGlnTyrGlyTyrCysGlyPheGlyAlaGluTyr                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      SerGlnTyrGlyHisCys                                                            15                                                                            __________________________________________________________________________

What is claimed is:
 1. A method for modulating the induction of an immune response to an antigen in a mammal by administering the antigen in combination with an effective amount of at least one lectin derived carbohydrate binding peptide capable of binding terminally linked α-sialic acid(2→6)βGal- and/or α-sialic acid(2→3)βGal- groups on structures or molecules comprising such groups wherein said lectin derived carbohydrate binding peptide is represented by formula I (SEQ ID No:1):

    SPX.sub.1 GX.sub.2 C                                       I

where X₁ is selected from the group consisting of amino acids Y, F, W, and H, and X₂ is selected from the group consisting of amino acids Y, F, R, W, and H.
 2. The method of claim 1, wherein said immune response comprises a humoral or cell mediated immune response.
 3. The method of claim 1, wherein said antigen comprises an allergen.
 4. The method of claim 1, wherein the dosage of said at least one lectin derived carbohydrate binding peptide ranges from about 0.5 to 50 mg/kg of body weight.
 5. A method for inducing, in a sensitized mammal, long term tolerance to an antigen by challenging said mammal with the antigen and followed by administering an effective amount of at least one lectin derived carbohydrate binding peptide capable of binding terminally linked α-sialic acid(2→6)βGal- and/or α-sialic acid(2→3)βGal- groups on structures or molecules comprising such groups wherein said lectin derived carbohydrate binding peptide is represented by the formula I (SEQ ID No:1):

    SPX.sub.1 GX.sub.2 C                                       I

where X₁ is selected from the group consisting of amino acids Y, F, W, and H, and X₂ is selected from the group consisting of amino acids Y, F, R, W, and H.
 6. The method of claim 5, wherein the antigen comprises an allergen.
 7. The method of claim 5, wherein the administration of said lectin derived carbohydrate binding peptide is about 1 to 10 hours after initiation of the mammal's inflammatory response to said antigen challenge but at or prior to one-half the period required for maximal inflammation to the antigen challenge.
 8. The method of claim 7, wherein the amount of the lectin derived carbohydrate binding peptide which is administered ranges from about 0.5 to 50 mg/kg of body weight. 